JPH0526470B2 - - Google Patents

Description

Claim 1: A monoclonal antibody having specificity for blood group antigenic determinants of human type B blood cells.
antibody). 2. The monoclonal antibody according to claim 1, which has specificity for an antigenic determinant of type B blood cells and is capable of causing agglutination of a type B blood cell sample having the determinant. 3. The monoclonal antibody according to claim 1 or 2, wherein the equilibrium constant of interaction between the antibody and type B blood cells is greater than 1.3×10 ⁷ M ⁻¹ . 4. The monoclonal antibody according to claim 3, wherein the equilibrium constant of interaction between the antibody and type B blood cells is greater than 1.2×10 ⁸ M ⁻¹ . 5. The single clone antibody according to claim 1 or 2, wherein the rate constant of the dissociation reaction of an immune complex containing the antibody and blood group B blood cells is smaller than 2.2×10 ⁻² sec ⁻¹ . 6. The monoclonal antibody according to claim 5, wherein the rate constant of the dissociation reaction of an immune complex containing the antibody and blood group B blood cells is smaller than 7.6×10 ⁻⁴ sec ⁻¹ . 7. The single clone according to claim 1 or 2, wherein the rate constant of the dissociation reaction of the immune complex containing the antibody and B blood cells of blood type _A1B is smaller than 5.0×10 ^-2 sec ^-1 antibody. 8. The monoclonal antibody according to claim 7, wherein the rate constant of the dissociation reaction of an immune complex containing the antibody and B blood cells of blood type _A1B is smaller than 3.4 x ^10-3 sec ^-1 . FIELD OF THE INVENTION The present invention relates to the field of biotechnology, and in particular to the use of monoclonal antibodies as blood grouping reagents. Background of the Invention When a foreign substance (antigenic substance) enters the body of a vertebrate, an immune response occurs. The purpose of this immune response is to prevent antigenic substances from causing damage to the animal's body and to eliminate such antigenic substances from the body. The immune system achieves this goal by producing immunoglobulin molecules (hereinafter referred to as antibodies) that have the property of selectively recognizing antigenic substances and binding to specific sites on the antigenic substance. These sites are known as determinants, and one antigen contains one such determinant.
It is possible to have one or more. Each antibody produced by the immune system has specificity for only a single determinant, but when the antigenic substance from which the antibody is produced has multiple determinants. several different antibodies are produced. The primary function of antibodies is to protect the body from harmful foreign substances by agglutinating them and thereby assisting the body's normal function of eliminating this material. Aggregation of antigenic substances by antibodies is also practically used outside the body in the field of blood group classification. Red blood cells have many different and characteristic antigenic determinants on their surface, and based on the nature of these determinants, blood can be divided into several types (e.g. A, B, O, A ₁ B, A ₂ B, B _cprd ). When blood is transfused from a blood donor to a blood donor, it is necessary that the transfused blood be of the same type as the blood of the blood donor. If they are not homotypic, the blood donor's immune system produces antibodies against the unfamiliar determinants on the surface of the transfused red blood cells. Such antibody responses to foreign red blood cells form the basis of blood typing techniques. In a broad sense, the classification of blood samples relies on the detection of red blood cell agglutination, etc., in the sample, which occurs when antisera directed against multiple determinants on the surface of red blood cells of that sample type are added. Agglomeration is a macroscopic effect that can be easily seen with the naked eye or automatically detected by a machine. Traditionally, the primary source of blood group separation reagents has been through hyperimmunization of human subjects. This is done by injecting into the subject's body a non-lethal amount of serum of a type different from the subject's blood type. This occurs during a normal immune response, resulting in the production of antibodies in the subject's blood. A sample of this blood is then taken and an antibody reagent is made from it. Such reagents may cause visible floeealation or aggin-tination of red blood cells if the unknown blood sample is of the same type as that which originally entered the body of the human serum donor.
can be used to classify unknown blood samples. In practice, there are two types of blood typing tests (F, Dunsford and See, Bowery, Blood Grouping Techniques, 2nd edition 1967). 1st
In this type of test, a sample of blood to be sorted is first placed on a blood grouping tile (see British Pharmacopoeia, section 'ABO donor determination'). This blood sample is then mixed with the antiserum sample on the tile. If agglutination subsequently occurs, it indicates that the unknown sample of blood to be classified belongs to the same blood type as the one for which the antiserum was produced.
In fact, such tile agglutination classification tests are routinely performed in emergency treatment. In the second type of test, the blood sample to be classified is placed in saline with the antiserum in a test tube and allowed to stand for a standard period of time (2 hours). The presence of flocculation can be determined by the sedimentation that occurs within a standard period of time. In emergency cases, centrifugation is used to expedite testing. The efficacy of a particular blood grouping reagent is determined by the rate at which it forms ag-glutinants and the manner in which its potency as an agglutinin is proportional to concentration. The first of these criteria is commonly referred to in the art as "avidity." The avidity time of a particular blood typing reagent is defined as the time required from mixing a blood sample with the reagent to when appreciable agglutination of the sample occurs. To determine the dilution properties of the blood grouping reagent, saline agglutination titers are measured. For this measurement, a plurality of equal volumes of a 2-fold diluted solution of the blood type classification reagent in physiological saline are prepared. A known amount of the appropriate red blood cell suspension is then added to each diluted solution. In this case add the same amount of suspension to all diluted solutions. Let each diluted solution sample stand for a standard time (usually 2 hours),
After that time, an agglutination count is performed under a microscope. Determine the highest dilution of the sample that results in a significant amount of agglutination and refer to this dilution as the “saline agglutination titer.”
title). Therefore, reagents with high saline agglutination titers are potent agglutinins. Two problems arise when using techniques of subject hyperimmunization to produce antibodies against erythroid determinants. First, serum is in limited supply, and as the frequency of many surgical procedures increases, the need for blood typing has increased significantly. For this reason, there is a shortage in the supply of human serum, which is also needed for other medical uses. Moreover, the agglutinating effect of antibodies on spontaneously produced red blood cells tends to be variable to some extent. One of the reasons is
The immune response is that each constituent antibody generates a "cocktail" of antibodies, each having a preferential effect on one determinant, as described above. Since it is not possible to separate the various antibodies in this cocktail, conventional antisera contain a mixture of antibodies that vary from animal to animal (and even within the same species and from day to day). ). Because there is no identical set of determinants for all red blood cells of the same blood type, immune responses are often highly variable. In summary, reagents suitable for blood grouping must meet the following requirements: (1) Must have specificity for an appropriate antigen. (2) be sufficiently powerful to show a good macroscopic response to relatively weak blood types, both in routine and emergency blood typing; (3) It must be stable under the conditions of use. (4) It should be relatively inexpensive and easily available. Recent developments in molecular biology have provided techniques for producing highly specific antibodies by producing hybrid cells (hybridomas) from antibody-producing splenocytes and myeloma cells. This technique, the work of Kohler and Milstein (Eur.J.
Immunol 6 292−295 (1976), Nature 256
495−497 (1975), Eur.J.Immunol 6 511−519
(1976)) provides a method for producing an infinite supply of antibodies. Because the antibody-producing hybrid cells produce only one type of immunoglobulin molecule, ie, an immunoglobulin with specificity for one determinant, the antibodies produced are termed monoclonal antibodies. Hybrid cells combine two desirable characteristics of myeloma cells and lymphoid cells. Thus, myeloma cells have immortal properties and can self-replicate in vitro, while lymphocytes have the desirable property of expressing antibodies. Such hybrid cells are therefore a permanent source of pure and defined immunoglobulins. The method used to produce hybrid cells is usually to immunize a mouse with the appropriate antigen, allow sufficient time for an immune response to occur, and then sacrifice the mouse and remove its spleen. . A cell suspension is then made from the spleen and this suspension is mixed with a suspension of mouse myeloma cells. Polyethylene glycol can be used to promote fusion of these two types of cells. As a result, a hybridoma culture obtained from one lymph cell produces one type of antibody, ie, an antibody specific for one particular determinant. This technique was used by Polk et al. (Vox Sanguinis 39 134-140 (1980)) to produce monoclonal antibodies against determinants of type A red blood cells.
Such a monoclonal anti-A antibody (monoclonal
anti-A') has been shown to be a useful blood grouping reagent. However, until now, it was generally believed that immunization of mice with human type B blood cells did not produce splenocytes that could fuse with myeloma cells to form hybridoma cells that produced anti-B immunoglobulin. Surprisingly, we found that this is not actually the case, and that fusion is easily obtained, resulting in the formation of hybridoma cells that express monoclonal anti-B antibodies with high efficiency, thereby providing beneficial saline solutions. It has been discovered that specific blood classification reagents of high avidity with agglutination titer properties can be produced. Furthermore, it has been found that the equilibrium constant and dissociation rate constant for the immune complex formed between the single clone anti-B antibody and type B blood cells can be defined. Since previously known blood grouping reagents contained mixtures of immunoglobulins with different specificities, such quantification of the power of immune complexes formed between blood grouping reagents and red blood cells was not possible. Analysis was impossible. Therefore, even the best figures that could be achieved hitherto were no more than average values. DESCRIPTION OF THE INVENTION In accordance with the present invention, monoclonal antibodies having specificity for antigenic determinants of type B blood cells are provided. As used herein, type B blood cells are meant to include all red blood cells that have one or more antigenic determinants found only on type B blood cells. Examples of blood types with such determinants are B, A ₁ B, A ₂ B and
B _cprd . Preferably, the monoclonal antibody is mouse, IgM, monoclonal anti-B immunoglobulin. Preferably, the monoclonal antibody has the ability to agglutinate a sample of type B blood having the above-mentioned determinants. A monoclonal antibody in which the equilibrium constant of interaction between the monoclonal antibody and type B blood cells is greater than 1.3×10 ⁷ M ^-1 , especially if this equilibrium constant is 1.2
It has been found that the above properties are particularly advantageous in the case of monoclonal antibodies larger than ×10 ⁸ M ^-1 . The values of equilibrium constants referred to herein are functional affinities.
affinities), or the interactions between multivalent antibodies (IgM) and red blood cells.
In this respect, this valence differs from the intrinsic affinities obtained for the interaction between a monovalent antibody (IgG) and an antigen. Intrinsic affinity is not directly comparable to functional affinity. The values that can be calculated for the monoclonal antibodies of the invention are dependent on the assay used and will be discussed in detail in the following description of the characterization of the monoclonal antibodies. Another feature of the present invention is that the monoclonal antibody has a rate constant of dissociation reaction of an immune complex containing the antibody and B blood cells of blood group B that is smaller than 2.2×10 ^-2 sec ^-1 , preferably It must be smaller than 7.6×10 ^-4 sec ^-1 . Another feature of the present invention is that the monoclonal antibody preferably has a dissociation rate constant of less than 5.0×10 ^-2 sec ^-1 for an immune complex containing the antibody and B blood cells of blood type A ₁ B. is smaller than 3.4×10 ^-3 sec ^-1 . The values of dissociation constants referred to herein are functional affinities as described above. The present invention further involves injecting a mouse with type B substance, sacrificing the mouse, removing its spleen to form a suspension of splenocytes, and fusing the splenocytes with mouse myeloma cells to generate hybrid cells. A method for producing a monoclonal antibody is provided, comprising: forming a monoclonal antibody, cloning the hybrid cell, and causing the cloned hybrid cell to secrete the monoclonal antibody. Preferably, said hybrid cells are selected prior to cloning. The myeloma cells are preferably NS1 cells. Furthermore, the present invention encompasses a monoclonal antibody produced by the above method, a hybridoma cell capable of secreting the monoclonal antibody, and a blood grouping reagent containing the monoclonal antibody. be. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the equilibrium binding between I-labeled anti-B antibody and type B red blood cells. Figure 2 is a modified Scatchard diagram showing the equilibrium binding of ¹²⁵ I-labeled anti-B antibody and type B red blood cells.
plot). FIG. 3 is a diagram showing the dissociation rate of I-labeled anti-B antibody from type B red blood cells at 4° C. and room temperature. Figure 4 shows 2×10 ⁶ red blood cells at room temperature or
FIG. 3 is a diagram showing the dissociation rate of I-labeled anti-B antibody from A ₁ B red blood cells. General description of hybridoma cell preparation Suitable mice were found by assaying serum samples for anti-B activity after absorption with type O blood cells to extract anti-species antibodies. Complete Freund's adjuvant (Difco Bacto) for mice with an anti-B agglutination titer of 1:8 after absorption.
100 μg of type B substance in 0.1 ml was injected intraperitoneally.
After 5 weeks, the same procedure was repeated, and after another 9 weeks, 200 μg of a 0.1 ml physiological saline aqueous solution of type B substance was additionally administered by intravenous injection. After 3 days, the spleen was removed and a splenocyte suspension was prepared. Splenocytes (10 ⁸ ) were fused with mouse myeloma NS1 cells (10 ⁷ ) using polyethylene glycol (Cotton et al. Eur. J. Immunol 3 135-140
(1973), Dunsford and Bowery (supra),
Galfre et al. Nature 266 550−552 (1977)). Growing hybrid cells were selected for their ability to produce specific anti-B activity. This was suspended in the culture supernatant by an agglutination assay using human A, B, O red blood cells. Anti-B-secreting hybrid cells were cloned twice on soft agar and finally grown in Dulbecco' Modified, Eagles, Medium (Dulbecco') supplemented with 5% v/v fetal calf serum (FCS, Sera-Lab). s Mobified Eagle′s Modium
(DMEM, Gibco Biocult)) in 1 liter of sp-inner-cultures.
Cloned hybrid cells were stored in liquid nitrogen. Cells and debris were removed by centrifugation, passed through a Millipore filter, and mixed with 10mM HEPES buffer.
Tissue culture supernatants containing monoclonal antibodies were prepared by adding 0.1% sodium azide. Aliquots of each batch were stored at 4°C for routine use and at -20°C for storage. Detailed description of materials and methods used for hybrid cell preparation Mice and rats B10, BR, C3H/He-mg and other mice were first obtained from OLAC.1976 Ltd. (Bicester, UK) and then OLAC mice. Medical Research Councii (Medical Research Councii)
obtained from stocks). AKR mice were obtained from Banting and King-man (York, UK). (C3H×BALB/C) _F1 mice and Lou, DA, AO rats received medical treatment.
Wistar, PVG, WAG and Sprague-Dawley rats, raised in Research, Council Animal Houses, were obtained from Bunting and Kingman. Mice for testing and immunization were 6-8 weeks old. ImmunizationHuman type B substances used for immunization Davrille, Dr. Watkins (Clinical, Research, Center, Harrow, United Kingdom), phenol (95
%), insoluble in ammonium sulfate (100%),
Human type B (and type A) is an extract of freeze-dried ovarian cyst fluid obtained by the Morgan method (1965), insoluble in ethanol (45-55%) and soluble in water. ) provided the substance. These contain 80-85% carbohydrates (L-glucose, D-galactose, N-acetyl-D-flucosamine,
N-acetyl-D-galactose), amino acid 15
~20% (mainly L-threonine, L-serine,
L-proline) and 1-2% sialic acid. These materials were received lyophilized, dissolved in 0.9% saline to 2 mg/ml, and stored at -20°C. B in complete Freund's adjuvant (CFA)
Preparation of type substance emulsion 1 ml of type B substance at a concentration of 2 mg/ml in 0.9% saline was prepared for Myco-bacterium tuberculosis with CFA (Difco Baeto), a mixture of Bayol F, oil and mannitutooleic acid detergent. mixture) was added to 1 ml. The mixture was vigorously stirred in two 5 ml syringes connected at right angles with a two-way tap (Warley, Chance, UK) and cooled intermittently on ice until a white creamy substance was formed (10-20 minutes). . This creamy substance did not spread even when dropped onto water. CFA remains one of the best immune response adjuvants (Bonford 1980
(see year). Because granulomas may form, repeated injections were performed at different locations. Immunization schedule To find suitable mice, blood was collected from the tail of unimmunized mice, absorbed with type O red blood cells to remove anti-species antibodies, and serum was assayed with type B red blood cells by in vitro agglutination. . 6-8 week old females with high anti-B titers
B10, BR mice and C3H/He-mg mice were injected intraperitoneally with one of the following three formulations:
That is, 100 μg of type B substance in 0.1 ml of CFA, 10 μg of type B substance in 0.1 ml of CFA, or 0.1 ml of packed type B red blood cells (washed 4 times). Two weeks later, blood was collected from the tails of the mice. B10, BR mice with high serum titers are given repeated intraperitoneal injections of 100 μg or 10 μg of type B substance every 5 weeks, or 3 times after the first injection.
0.1 ml of red blood cells after 1 week, 4 weeks and 5 weeks
was injected repeatedly. Mice were bled from the tail one week, three weeks and six weeks after the last injection. Collection of serum Collect 0.5 ml of blood from the mouse tail into a synthetic resin tube, leave it at 37°C for 1 hour to form a clot, and
The tube was released to help deflate the clot. Dilute the separated serum with 0.9% physiological saline,
The cells were incubated in a 56°C water bath for 20 minutes and absorbed with O blood cells or _A1 blood cells as necessary. Aliquot at −20°C
It was stored in Preparation of monoclonal antibodies against blood group B The general outline of the process at this stage is based on the basic principles written by Kohler and Milstein (see above) for producing monoclonal antibodies with the desired reactivity. (see literature). Splenocyte hypoxanthine guanine phosphoribosyl transferase (HGPR-Tase)
+ve specific Ig +ve terminally differentiated myeloma cell HGPRTase −ve specific Ig −ve immortal −ve hybrid cell HGPRTase +ve specific Ig +ve immortal Myeloma cells that are not fused lack HGPRTase; therefore it dies in the chosen medium (Littlefield, Science
145, 709−710, 1964). Also, unfused splenocytes cannot survive in tissue culture. Only hybrid cells of splenocytes and myeloma cells have HGPRTase.
and immortality, and therefore able to survive. Cells secreting antibodies with the required specificity can then be selected. That is, a hybrid cell has the respective strengths of both parent cells. Stock solution 50% polyethylene glycol (PEG) solution 10 g of PEG1500 (BDH Lot G573370) was autoclaved and immediately transferred to a 45°C water bath and then at 37°C.
10 ml of DMEM was added. Aminopterin Stock 1000X Aminopterin (Sigma) slightly warmed
Dissolved at 0.176mg/ml in 0.008M NaOH at -20°C
It was stored in the dark. HT Stock 100X 136.1 mg of hypoxanthine (Sigma) and 38.75 mg of thymidino were normally dissolved in 100 ml of DDW and stored in 50 ml aliquots at -20°C. 50ml DMEM 50ml HT100X The prepared solution was filtered (Millipore, pore size
0.22 μm) stored in 13 ml aliquots at -20°C.
After thawing, the HT was heated at 60-70°C to re-dissolve it. HAT medium 50X DMEM 45ml HT100X 50ml Aminopterin 1000X 5ml The solution was divided into 13ml aliquots as above.
Drained, stored and thawed. HAT in 20% FCS-DMEM (HAT Vehicle) DMEM 400ml FCS (Batch 901112) 100ml P/S 8ml Glutamine 8ml Pyrimidine (Pyr) 6ml HAT Media 50X 10.6ml (1.0 x 1.0 ^-4 M Hypoxanthine, 4.0 x 4.0 ^{- 7} M aminopterin, 1.6 x 10 ^-5 M thymidine) HT in 20% FCS-DMM (HT vehicle) HAT50X in the above preparation method was replaced with 10.6 ml of HT50X. Therefore, we will use the following terms: Serum-free DMEM (serum froo DMEM) = DMEM
+PS + Glutamine + Pyrimidine 20% FCS-DMEM = Serum-free DMEM + 20% FCS HAT medium = 20% FCS-DMEM + HAT HT medium = 2% FCS-DMEM + HT Production and initial selection of anti-B myeloma hybrid cells The method described by Kohler et al. Cell fusion was performed according to (see above-mentioned literature). In preparation,
The following media were equilibrated overnight at 37° C. in a 5% CO ₂ atmosphere in air. (1) 50% PEG solution (2) Serum-free DMEM (3) 20% FCS-DMEM The above medium was prepared in 48 Linbro wells.
Contains 1.5 ml aliquots distributed into wells). On the morning of the fusion, the spleen was removed under sterile conditions.
Cells were aliquoted into 10 ml of serum-free DMEM and then transferred to a 10 ml centrifuge tube. A period of 5-10 minutes was allowed to settle the cell clumps, and then the suspended cells were pipetted out and washed twice in serum-free DMEM. 1
1.4 x 10 ⁸ suspended cells were obtained from each spleen. ¹⁰⁸ of these cells were collected in 5-10 ml of serum-free DMEM. At the same time, 2.6× in spina culture
10 ⁷ myeloma NSI cells cultured at 10 ⁶ /ml were washed twice and resuspended in 5 ml of serum-free DMEM. The medium containing splenocytes (10 ⁸ ) and myeloma cells (10 ⁷ ) was pooled in a 50 ml Corning tube, and the cells were pelleted by centrifugation at 400 g for 5 min at room temperature, followed by a Pasteur pipette with an aspirator pump. to aspirate the medium as completely as possible. Support the tube in a beaker of 37°C water and add 50% PEG solution (equilibrated at 37°C).
1 ml was added slowly over 1 minute followed by gentle stirring with the end of the pipette used for this addition for an additional minute. The suspension was then diluted with serum-free DMEM (37
℃ equilibrated) at 1 ml/min for the first 2 minutes,
The suspension was diluted at a rate of 2 ml per minute for the next 4 minutes, then 10 ml dropwise and then slowly added at will to 50 ml. The tube was gently agitated after each addition. Centrifugation (5 minutes at 400g)
Thereafter, the cells were suspended in this solution by gently injecting them into 25 ml of 20% FCS-DMEM (in parallel) using the 24 ml pipette used for the addition. The fusion mixture in 20% FCS-DMEM was then distributed in 0.5 ml aliquots into 48 parallel wells containing 1.5 ml of 20% FCS-DMEM. As feed cells, the remaining splenocytes were diluted and added dropwise at a rate of approximately 4×10 ⁵ /well. The culture tray was then exposed to 5% _CO2 ,
Incubation was carried out at 37° C. in a 45% temperature atmosphere. The next day (day 1) and the 2nd and 3rd days after cell fusion
On days 1, 7, and 11, the top half of each culture medium was removed (separate Pasteur pipettes were used for each well to avoid possible cross-contamination) and incubated with 5% _CO2 in air. An equal volume of equilibrated HAT selection medium (Littlefield 1964) was substituted. Cultures were observed daily to check for hybrid cell growth and possible yeast or microbial contamination. At this time, the time the culture medium was left outside the incubator was kept as short as possible. When cell monolayer growth approaches full growth (approximately 14 to 21 days after cell fusion)
On day 1, which is approximately the same time when the medium changes color from red to yellow (phenolic), the culture supernatant was tested for the first time (by agglutination with type A, type B and type O red blood cells). On that day, positive cultures were subsorted by resuspending the cells with a 1 ml pipette and transferring 1 ml of the cell suspension to 1 ml of HAT medium (equilibrated at 37°C). The original culture was supplemented with HAT medium. As a safety measure against incubator failure, split cultures were grown in separate incubators, periodically assayed by aggregation, and frozen in liquid _N2 in FCS containing 10% DMSO as soon as possible. Negative cultures were retested when the medium appeared yellow. Contamination with yeast or microorganisms was addressed by adding 45% EtOH, aspirating the culture for 5 minutes, and washing the empty wells with EtOH. Aliquoted media on culture trays were always aspirated immediately. Subcultures were retested 4-10 days after the initial testing of the fusion wells, and aliquots of positive cultures were frozen or diverted for cloning. Isolation of anti-B-secreting hybrid cells Cloning of cells from selected cultures was performed on soft agar as described by Cotton et al. (see above). 1% in 0.9% saline
HAT− agar (Difco Bacto) 50ml (45℃)
DMEM2X (2X DMEM100ml + p/S4ml + Glutamine 4ml + Pyridine 4ml + FCS75ml + 50X
HAT 7.8ml) (45℃)
A 0.5% agar solution in HAT medium was prepared. This agar mixture was placed in a 9cm Petri dish (N-unc).
15 ml of the mixture was pipetted into each dish, allowed to solidify for 5 minutes in a laminar flow tissue culture hood with the lid off, and then equilibrated for 30 minutes in a gas incubator. Add 0.5% agar HAT-DMEM1 to 1 ml of cells in 6 cups of 3x DMEM solution prepared in a phosphorus well.
ml was added. 2 ml of the mixture was layered evenly on the set agar, air-dried as above, and dried in the air for 5 minutes.
Incubated at 37°C in an atmosphere of % _CO2 . After 14-20 days, multiple discrete macroscopic cell colonies were formed. Many of them grew from single cells. These colonies were picked using a fine Pasteur pipette from the dish containing the minimum number of colonies (usually 10-20) and added to 1 ml of HAT-DMEM equilibrated in a gas-filled incubator.
Planted in a linbrow well containing. Following normal cycles of culture growth, the cultures were examined for the presence of aggregation, and some were frozen in liquid _N2 . Half of the culture was then diluted with an equal volume of
Transfer to HT media 2 or 3 times and then 20%
Through FCS-DMEM, a total of 5
HAT selected clones by taking ~6 days
I pulled away from it. Cells were harvested as described above, but with 0.5% agar.
Recloned in 20% FCS-DMEM. Cells thus cloned twice were selected on the basis of (1) the ability of the culture supernatant to exhibit strong agglutination of red blood cells and (2) rapid high-density cell growth. Every 2 days, half of the culture was added to 10%, 5%,
Selected clones were allowed to grow in low FCS by transferring each to medium containing 2.5% FCS. Cloning by limiting dilution and adding feed cells This method was used for hybrid cells that did not grow well when transferred to agar at low density. The cell suspension was diluted to 1:32 in Linbrow wells as a doubling dilution series, and several series of these were prepared. To each row 100 μ of growth medium was added containing each of the following as feed cells: (1) 4×10 ⁵ B10, BR thymocytes (2) 4×10 ⁵ B10, BR splenocytes (3) 4×10 ⁵ X-ray irradiated 3T3 mouse fibroblasts (20000 rads at 1 rad/sec) (4) 4 ×10 ⁵ B10 treated with mitomycin ^* ,
BR thymocytes (5) 4 x 10 ⁵ B10 treated with mitomycin ^* ,
BR splenocytes (6) None Hybrid cells were compared at low dilutions to determine their suitability for collecting isolated colonies. *Add 0.1 ml of mitomycin C (Sigma) at 0.5 mg/ml in EBSS to 1 ml of medium containing 10 ⁷ cells;
Incubation was performed at 37°C for 30 minutes and washing was performed three times. Storage of hybrid cells in liquid _N2 Frozen stocks were prepared from exponentially growing cells from 2 to 8 x ¹⁰⁵ cells/ml. Cells pelleted from 10 ml of culture supernatant were resuspended in 2 ml of 90% FCS-10% DMSO (ie approximately 10 ⁶ /ml) and then divided into two sterile cryobottles (10 ⁶ per bottle). Each bottle was cooled on ice for 2-4 hours and then overnight in the vapor phase of a Linde liquid _N2 tank.
Finally immersed in liquid _N2 . This operation cools the cells to about 1° C./min. To grow cells from frozen stocks, thaw the cryobottle in a 37°C water bath and pour the contents into 10ml of growth medium.
It was slowly diluted inside. Precipitated cells (400g,
5 minutes) was resuspended in 5 ml of 5-10% FCS-DMEM. Prepare 10 ⁷ bottles of cells from the spinner culture for an immediate start of 50 ml. Example of naming antibody-secreting hybrid cells Anti-B producing clones NB1/19, 112, 28 Each hybrid cell will be named by the name related to the cell fusion experiment. In the above case, N of NB1 is parent myeloma NS1
and B indicates anti-B splenocytes. The first number (19 in the example above) is the first uncloned culture well from which the clone was obtained.
well). Subsequent numbers (112 and 28) indicate the respective culture wells in which selected clones were grown following the first and second agar cloning. The number of culture wells in one phosphorus tray is 1~
Add 24 numbers or letters A1 to 1D6. Therefore, if 5 trays are used, the wells will be 1 to 120 or 1A1 to 5D6. The entire designation indicates the cell clone, the monoclonal antibody produced or its antigen specificity. Characterization of single clone anti-B Three stable tissue culture lines of cloned anti-B producing hybrid cells (NB1/19.112.28, NB1/
6.36.36 and NB1/48.30.40) were obtained by fusion between splenocytes of mice challenged with type B antigen and a mouse myeloma lineage. Tissue culture supernatants containing secreted monoclonal antibodies were tested separately for the three series. Blood groups taken from the same batch
References Laboratory (Blood Group
The efficiency of monoclonal anti-B antibodies produced by hybridoma cells was assessed using a human anti-B sample (BGRL No. 7327) from a commercially available human anti-B sample from a commercially available commercially available human anti-B sample. Hemagglutination tests were performed according to the method of Dasford and Bowery (supra). Red blood cells are ACD
or washed four times with a 7-day old clot sample (from the Regional Blood Trans-fusion Center, Cambridge) before use. A 20% red blood cell suspension in saline was used for the tile test and a 2% suspension was used for the standard 2 hour tube gravity sedimentation test. 2% papain-treated cell suspension or 20% bovine albumin was used for enhancement tests. Antibody dilutions were made in saline. In a standard 2-hour tube sedimentation test, fully grown culture supernatants exhibited the anti-B agglutination titers shown in Table 1. [Table] The differences between the three monoclonal antibodies described above were semi-reversible in the precipitation test described above and other tests described above, with antibody NB1/19.112.28 consistently showing superior results. As expected, A ₁ B adult blood cells and Bcord blood cells showed somewhat lower titers than adult A ₂ B blood cells and adult B blood cells. This is because A ₁ B adult blood cells and Bcord blood cells are weak type B blood cells. All reagents tested showed sufficient agglutination for all blood types on the panel, but NB1/
The activity of 19.112.28 was significant against the six weak Beord blood types shown in the table. 5 different types
A ₁ B for blood (not shown in table)
The activity range of NB1/19.112.28 is similar to that of BGRL anti-B.
16.32 (mean 32) versus 256−1024 (mean titer 512)
It was hot. In further experiments, NB1/48 and
The saline agglutination titer of the purified product of NB1/19 was determined on 2% type B blood cells and 2% A ₁ type B blood cells. The purified NB1/48 and NB1/19 fractions were mi-erofugeed and A ₂₈₀ = 1.00 (i.e. 100 μg/ml) with A1% 1 cm and 280 = 10.
)
Add each fraction to PBS (phosphate buffered
saline). Ammonium sulfate cuts of the culture supernatant (from which fractions were purified) were microfuged and diluted 1/16 in PBS. Serial 2-fold diluted solutions were prepared at a pH of 7.4 by pipetting with a Hamilton syringe and washing the syringe four times between each pipetting movement.
Made in PBS. A 25 μ aliquot of each diluted solution was incubated in glass tubes with 25 μ (5×10 ⁶ ) of 2% red blood cells suspended in PBS at PH 7.4 for 2 hours at room temperature. The cell pellet was carefully transferred to a glass slide and the number of aggregates counted. The culture supernatants used in this experiment are those shown in Table 1 above. The results are shown in Table 2. [Table] Meaning of symbols in Table 2 C: complete agglutination V: very strong agglutination ++) +) (+): moderate agglutination GW: somewhat weak W: weak In the tile agglutination test that reflects antibody binding activity, The relatively weak reaction between Bcerd blood cells and adult A ₁ B blood cells more clearly demonstrated the difference in anti-B activity of various different reagents compared to adult B blood cells and adult A ₂ B blood cells.
The results are shown in the third section. [Table] A ₁ Single clone anti-B NB1/19.112.18 against B blood cells and B _cprd blood cells can be found in commercial reagents within seconds when used without concentration and without additives. produced a strong agglutination reaction similar to that of
Although the BGRL reagent produced satisfactory macroscopic results, the rate and extent of aggregation were inferior. In further experiments, 20% B red blood cells and 20% A ₁ B red blood cell NB1/48 and
Tile aggregation time by NB1/19 was measured. The reagents used were the same as described in connection with Table 2, except that red blood cells were used at 20%. A 2μ aliquot of each dilution was mixed with 20μ of red blood cells on an opaque glass tile and shaken regularly. A pedal-operated stopwatch was started at the moment of mixing. Table 4 shows the time (seconds) at which aggregation occurs.
and shows the degree of aggregation after 5 minutes. Table 3 shows the degree of aggregation.
It is the same as defined in relation to . For example, 13V indicates that ten aggregation occurred after 13 seconds and very strong aggregation occurred after 5 minutes. [Table] [Table] The results obtained for 2-fold diluted solutions of different reagents and the relative potency of NB1/19.112.28 are shown in Table 5. For example, at a 4-fold dilution, the culture preparations tested showed satisfactory responses to A ₁ B blood cells compared to commercially available anti-B blood cells. [Table] Not all monoclonal antibodies with ABO specificity are equally suitable for blood group classification. In the example given here, three single clone anti-B
Although two of them had properties comparable to the BGRL standard, they were poor blood grouping reagents compared to the other one. The specificity of single clone anti-B allows for the determination of defined functional affinities. Functional affinity can be expressed in two ways. First, functional affinity is
It can be expressed as the equilibrium constant of the association reaction between a single clonal anti-BI _g M molecule and a B determinant (of the corresponding immunochemical type) on red blood cells. This equilibrium constant (hereinafter referred to as K) is obtained by first measuring the equilibrium binding amount of ¹²⁵ I-labeled anti-B antibody and type B red blood cells at different levels of binding-capable antibody present in solution. The experiment was conducted as follows. All measurements were performed in duplicate. ¹²⁵ I-labeled NB1/
19 and NB1/48 linear dilution series (linear
buffer (0.8% BSA-
Conditioned in EBSS + 10mM Hepes + 0.1% NaN ₃ PH7.4). A 25 μ portion of ^{the 125} I-labeled antibody was placed in a 1.5 ml Beckman microfuge tube (Beckman
microfuge tubes) and then 15 μ of buffer containing 2×10 ⁶ fresh type B blood cells were added. The measuring mixture was incubated on a roller at 4°C. 6
After an hour, 1.5 ml of ice-cold buffer was rapidly added, and the microfuge was immediately rotated for 15 seconds to remove the supernatant. The time from adding the buffer to turning on the microfuge was 2 to 4 seconds. In control incubation,
Replace type B blood cells with 2 x ¹⁰⁶ type _A blood cells, add 1.2-2.0% and 0.9-1.2% of the possible binding counts, respectively.
The binding of ¹²⁵ I-labeled NB1/19 and NB1/48, which accounted for 125 I, was subtracted from the experimental values. The results are shown in FIG. FIG. 1 is a plot of the amount of ¹²⁵ I antibody bound (epm×10 ^-3 ) against the amount of antibody that can be added and bound for NB1.19 and NB1/48. From these graphs, a modified scattering plot of the equilibrium binding properties of ¹²⁵ I-labeled anti-B to type B red blood cells can be constructed. Such a diagram is shown as FIG. Figure 2 shows values for both NB1/19 and NB1/48. NB1/48 is shown enlarged in the inner graph. In both cases, extrapolation to the X-axis indicates the amount of bound antibody at saturation. The equilibrium constant can be easily calculated from a scattering plot. Functional affinity is also expressed as the dissociation rate constant of the dissociation reaction of the immune complex formed between a single clonal anti-BI _g M molecule and the B determinant (of the corresponding immunochemical type) on red blood cells. You can also do that. The dissociation rate constant (hereinafter referred to as K-1) is 0
It can be calculated from a graph of the change in dissociation time of ¹²⁵ I-labeled single clone anti-B from red blood cells by measuring the slope of the tangent to the dissociation curve formed over time. In one experiment, the rate of dissociation of ¹²⁵ I-labeled anti-B antibodies from type B red blood cells at 4°C and room temperature was determined in the presence of an excess of chilled antibody. ¹²⁵ I label NB1/19
(Bindable antibody: 6.0×10 ⁵ cpm, 50ng)
NB1/48 (9.0×10 ⁵ epm: 124n _g ) 1.5 in 25μ increments
ml microfuge tube and then type B red blood cells 2
Buffer containing × ¹⁰⁶ (0.8% BSA-EBSS + 10mM
25 μ of Hepes + 0.1% NaN ₃ PH7.4) was added. The mixture was incubated on a roller at 4°C. After 1 hour, add ice-cold buffer to 4 of these tubes.
Immediately after rapidly adding 1.5 ml of blood cells to an Eppendorf mierofuge
The mixture was spun for 15 seconds in a vacuum chamber to allow precipitation. The supernatant was quickly removed and blood cells were transferred for measurement of bound radioactivity (epm). To the remaining measuring tube after incubation for 2 hours.
A 125-fold concentrated ammonium sulfate solution of the NB1/19 culture supernatant was diluted to 1/10 with a buffer solution and 25 µl of the solution was added. 75μ of the mixture was then incubated on a roller at 4°C or room temperature. Different times (15 minutes ~
After 24 hours, a cold buffer was added to precipitate the blood cells immediately and counted as described above. In the control incubation, 2 × 10 ⁶ type A ₁ blood cells were replaced with type B blood cells, and after incubation for 1 hour at 4°C,
Combination of NB1/19 and NB1/48 (additional count of 1.1
% and 1.9%) were subtracted from the experimental values. Binding was therefore expressed as a percentage of the number bound at the end of the 2 hour pre-incubation period. ¹²⁵ I label NB1/19
and 100% binding at NB1/48 is 9.1×, respectively.
It was ¹⁰⁴ epm and 715× ¹⁰⁴ . In the final reaction mixture, at least a 40-fold excess of the labeled antibody, considering the concentration of monoclonal antibody in the culture supernatant to be at least 10 μ/ml, was added to the cold BN1/ml.
There were 19. The above results are shown in FIG. Figure 3a shows the dissociation over a 24 hour period and Figure 3b is a replot of the first 45 minutes with an expanded time axis. In yet another experiment, the rate of dissociation of ¹²⁵ I-labeled anti-B antibodies from 2 x 10 ⁶ type B blood cells or A ₁ type B red blood cells at room temperature was determined in the presence of an excess of chilled antibody. The experimental procedure was similar to that described above, except that after adding the chilled antibody to the antigen-labeled antibody complex, the blood cells were allowed to precipitate every 2 minutes and continued for 10 minutes. The resulting processing is the same as above. As a result, the following values were used or obtained. [Table] The results are shown in Figure 4. Figure 4 relates to type B red blood cells, and Figure 4 relates to type A ₁ B red blood cells. The functional affinities measured in each of the experiments described above and the properties measured in the other experiments described above are summarized in Tables 6 and 7 below for NB1/19.112.18 and NB1/48.30.40. [Table] [Table] [Table] Finally, the efficacy of NB1/19.112.28 as a blood grouping reagent when used in an automatic blood grouping machine,
NB1/when using papain-treated red blood cells
Some experiments were performed to test the selectivity of 19.112.28 and its thermal stability. Automated blood grouping tests were performed using a modern automated blood grouping machine 16C with optical density reading and microprocessor printout capabilities. The reagents were then enriched with 1% methylcellulose and 0.25% primeline. A 1/15 dilution of the monoclonal antibody NB1/19.112.28 culture supernatant was tested on an automated blood typing machine 16C on 844 citrated donor blood assays.
The results shown in Table 8 are excellent;
The results were identical to those obtained using the B1/10 dilution on the same sample. Neither reagent gave false positive or negative results. [Table] In the above in vitro and tile saline tests and automated tests using blood cells enhanced with 1% methylcellulose and 0.25% promeline, single clone anti-B did not interact with A type ₁ blood cells or type 0 blood cells. I didn't react well. N1B/
The anti-B specificity of 19.112.28 is as shown in Table 9.
It was also maintained when treated with papain and when using 20% albumin. NB1／
19.112.28's marginal enhancement effect on aggregation activity (marginal
enhancement) was observed in these tests, especially for papain-treated _A1B blood cells. [Table] In the stability test, undiluted NB1/19.112.28
Titer and binding were measured again at room temperature after exposing 2 ml aliquots of culture supernatant to different conditions. Repeated freezing and thawing (see Table 10) did not reduce anti-B activity levels. [Table] * Dilution after processing of undiluted supernatant. Short-term thermal stability studies (see Table 11) showed that single clone anti-B was stable for storage at -20°C and 4°C, as well as for storage at 56°C. It has been shown that it is stable even with incubation at . [Table] * Dilution of undiluted supernatant after processing undiluted supernatant has at least the same potency as the hyperimmune commercial reagent without the use of additives. It exhibits a rapid, tightly aggregated tile reaction suitable for detecting weak type B (A ₁ B and B _cprd blood cells) encountered in routine classification tasks. It can also be safely used for automatic blood grouping. Twice-cloned antibody-producing hybrid cell lines are adapted to grow in large tissue culture vessels and continue to secrete antibodies of uniform properties through repeated passages. The large quantities of active culture supernatants produced by continuous growth will require a number of screening tests far greater than is currently required to produce the same amount of human reagent from a large number of individually screened donor sera. It has the advantage that it requires less time and the effort required for testing can be significantly reduced. The cost of materials to make potent culture supernatant 1 is also as low as £5, but this cost can be further reduced by using acceptable dilution preparations of the active supernatant (see Table 5). can do.